The tropism of Salmonella for dendritic cells and macrophages in the lymphoid tissue of the intestinal mucosal surface, naturally targets attenuated derivatives of this organism to these inductive sites and allows for the exploitation of this property in the development of a mucosal HIV- 1 DNA vaccine. Accordingly, we and others have recently reported that genetically-engineered, attenuated Salmonella strains are capable of delivering DNA vaccines to mouse tissues where they elicit humoral and cell-mediated immune responses against passenger antigens encoded by the DNA vaccine. Preliminary data generated in our laboratory also suggests that a single oral dose of a Salmonella HIGV-1 Env DNA vaccine induces similar numbers of interferon-gamma-secreting CD8+ T cells as those induced by the naked Env DNA vaccine injected intramuscularly as a single mug dose. The principle objective of this project is to obtain preclinical data on the safety and immunogenicity of HIV-1 Env DNA vaccines delivered by attenuated Salmonella in animal models. This data is required by Project 3 to test the principal hypothesis on our IPCAVD program that such vaccines are safe and immunogenic in human volunteers. We will obtain this data in the following specific aims: 1) to construct and characterize a prototype attenuated Salmonella typhi HIV-1 gp120 DNA vaccine vector: We will construct two HIV-1/Ba-L gp120 DNA vaccines and introduce them into our attenuated Salmonella typhi vector strain, CVD DELTA/ASD. The first will be comprised of a gp120 DNA vaccine of the HIV-1-Ba-L isolate (gp120Ba-L). This gp120 has been codon-optimized for expression in mammalian cells. The second will be compromised of a chimeric gene encoding a gp120-CD4 fusion protein that is expected to prime for broadly neutralizing antibodies against HIV-11 (see also Project 2); and 2) to obtain preclinical safety, immunogenicity and genetic toxicity data on the Salmonella HIV-1 DNA vaccines developed in aim 1. In the second phase of this study we will obtain preclinical safety, immunogenicity and genetic toxicity data on the Salmonella HIV-1 DNA vaccine vector constructs. Although Salmonella typhi is only fully virulent in humans, murine models have been developed for the assessment live lymphoid vaccine safety and immunogenicity. The information gained from these studies will be used in IND applications to conduct Phase 1 safety and immunogenicity studies in volunteers as described in Project 3.